retrofit of zeiss axiovert and lsm 510 for shg imaging

[Owens, Peter]

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Dear all,

I would like to use a ziess axiovert  microscope (LSM 510 confocal unit)
coupled to a spectra physics tsunami multiphoton laser to image the shg
signal from collagen fibres in heart valves.  

I am wondering what is the best way to do this and can anyone give
comments / recommendations on how our  existing machine may be
retrofitted for this task.

Thanks

Peter Owens


__________________________________
Dr. Peter Owens
Centre for Microscopy and Imaging and NBIPI
National University of Ireland Galway
Landline: 0035391494036
Mobile:    [phone]
------------------------------------------------------

 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Mark Cannell
Sent: 02 May 2012 17:21
To: [hidden email]
Subject: Re: confocal PMT intermitently dies

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Check connectors...  Unplug and replug all connectors. If that doesn't
work then its most likely electronic in the pmt3 power supply or pmt3
preamp stages...

Hope this helps
Mark

On 2/05/2012, at 4:22 PM, Arvydas Matiukas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
>
> Ch3 PMT intermitently dies on our LSM510 system. After 2-4 hrs of
> powering on, all image pixels turn to  zero intensity no matter what
> are laser power, pinhole, filter, and gain settings are used.
> Cold reboot does not fix the problem though no error messages are
> reported. The single fix so far is to let the system cool down for
> several hours then it is again working for few more hours.
> Surprisingly all cooling fans are spinning normally as well as  room
> conditioning system.
>
>
>
>
> We are off service contract so any feedback/advice what could be wrong

> and how to fix it is greatly appreciated.
>
>
> Thank you,
> Arvydas
>
>
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Imaging Core Facility Department of
> Pharmacology SUNY Upstate Medical University
> 766 Irving Ave., WH 3159
> Syracuse, NY 13210
> tel.: [phone]
> fax: [phone]
> email: [hidden email]

[Nuno Moreno]

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Hi,

At a certain point he had our 510 with SHG capability. In practice you need to direct couple the laser (one inch northwest from the visible laser fiber you should have a hole), change some dichroic mirrors and ask a quote for a PMT on the forward detection. Alignment is pretty easy by using a visible laser reflection. There might be further details though, namely the ms access db configuration.

Do not expect a good performance...we had better signal with http://www.sciencedirect.com/science/article/pii/S0968432804000897  than with the zeiss, both forward and backward direction.

Have a look at the objectives specs. Don't forget that you might be imaging at 800 and collecting at 400.

All the best,
NM

PS: Don't forget
http://www.facebook.com/CoreManagementWorkshop



On May 3, 2012, at 3:30 PM, Owens, Peter wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I would like to use a ziess axiovert  microscope (LSM 510 confocal unit)
> coupled to a spectra physics tsunami multiphoton laser to image the shg
> signal from collagen fibres in heart valves.  
>
> I am wondering what is the best way to do this and can anyone give
> comments / recommendations on how our  existing machine may be
> retrofitted for this task.
>
> Thanks
>
> Peter Owens
>
>
> __________________________________
> Dr. Peter Owens
> Centre for Microscopy and Imaging and NBIPI
> National University of Ireland Galway
> Landline: 0035391494036
> Mobile:    [phone]
> ------------------------------------------------------
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Mark Cannell
> Sent: 02 May 2012 17:21
> To: [hidden email]
> Subject: Re: confocal PMT intermitently dies
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Check connectors...  Unplug and replug all connectors. If that doesn't
> work then its most likely electronic in the pmt3 power supply or pmt3
> preamp stages...
>
> Hope this helps
> Mark
>
> On 2/05/2012, at 4:22 PM, Arvydas Matiukas wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear list,
>>
>>
>> Ch3 PMT intermitently dies on our LSM510 system. After 2-4 hrs of
>> powering on, all image pixels turn to  zero intensity no matter what
>> are laser power, pinhole, filter, and gain settings are used.
>> Cold reboot does not fix the problem though no error messages are
>> reported. The single fix so far is to let the system cool down for
>> several hours then it is again working for few more hours.
>> Surprisingly all cooling fans are spinning normally as well as  room
>> conditioning system.
>>
>>
>>
>>
>> We are off service contract so any feedback/advice what could be wrong
>
>> and how to fix it is greatly appreciated.
>>
>>
>> Thank you,
>> Arvydas
>>
>>
>>
>>
>>
>> Arvydas Matiukas, Ph.D.
>> Director of Confocal&Two-Photon Imaging Core Facility Department of
>> Pharmacology SUNY Upstate Medical University
>> 766 Irving Ave., WH 3159
>> Syracuse, NY 13210
>> tel.: [phone]
>> fax: [phone]
>> email: [hidden email]

[Armstrong, Brian]

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Peter, you should be able to do SHG with the set-up you have without changing anything. It will be in the "reverse direction" and as Nuno stated "do not expect good performance".
If you have the NDD system then you should use one of the NDD configurations and do this in complete darkness (I turn off all lights, drape the scope, and turn off the computer monitors).

If this does not suffice; then updating the LSM510 in order to perform good SHG imaging may not be worth the expense and headache (just my opinion).

Best of luck.  

Brian D Armstrong PhD
Assistant Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Owens, Peter
Sent: Thursday, May 03, 2012 7:31 AM
To: [hidden email]
Subject: retrofit of zeiss axiovert and lsm 510 for shg imaging

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear all,

I would like to use a ziess axiovert  microscope (LSM 510 confocal unit)
coupled to a spectra physics tsunami multiphoton laser to image the shg
signal from collagen fibres in heart valves.  

I am wondering what is the best way to do this and can anyone give
comments / recommendations on how our  existing machine may be
retrofitted for this task.

Thanks

Peter Owens


__________________________________
Dr. Peter Owens
Centre for Microscopy and Imaging and NBIPI
National University of Ireland Galway
Landline: 0035391494036
Mobile:    [phone]
------------------------------------------------------

 

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Mark Cannell
Sent: 02 May 2012 17:21
To: [hidden email]
Subject: Re: confocal PMT intermitently dies

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Check connectors...  Unplug and replug all connectors. If that doesn't
work then its most likely electronic in the pmt3 power supply or pmt3
preamp stages...

Hope this helps
Mark

On 2/05/2012, at 4:22 PM, Arvydas Matiukas wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
>
> Ch3 PMT intermitently dies on our LSM510 system. After 2-4 hrs of
> powering on, all image pixels turn to  zero intensity no matter what
> are laser power, pinhole, filter, and gain settings are used.
> Cold reboot does not fix the problem though no error messages are
> reported. The single fix so far is to let the system cool down for
> several hours then it is again working for few more hours.
> Surprisingly all cooling fans are spinning normally as well as  room
> conditioning system.
>
>
>
>
> We are off service contract so any feedback/advice what could be wrong

> and how to fix it is greatly appreciated.
>
>
> Thank you,
> Arvydas
>
>
>
>
>
> Arvydas Matiukas, Ph.D.
> Director of Confocal&Two-Photon Imaging Core Facility Department of
> Pharmacology SUNY Upstate Medical University
> 766 Irving Ave., WH 3159
> Syracuse, NY 13210
> tel.: [phone]
> fax: [phone]
> email: [hidden email]

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[Pascal Weber]

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What kinds of comments you want? how to align the IR laser!
What will make the SHG? To align the IR laser desserts you a visible laser
reflection in a mirror you will see out through the IR input beam visible. All you
need to align two pinholes and convey your IR laser by two holes. For the SHG
detectors you can use the simple but their performance will be low. The SHG is
always half the wavelength of excitation. The best way is ti use  NDD

[Craig Brideau]

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Can you give us a bit more detail regarding your setup?  For instance, do
you already have an appropriate dichroic?  Do you know what beam diameter
the laser has to be when it enters the scan head?  Have you done any
modification like this before or are you going in cold?

Craig



On Thu, May 3, 2012 at 8:30 AM, Owens, Peter <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear all,
>
> I would like to use a ziess axiovert  microscope (LSM 510 confocal unit)
> coupled to a spectra physics tsunami multiphoton laser to image the shg
> signal from collagen fibres in heart valves.
>
> I am wondering what is the best way to do this and can anyone give
> comments / recommendations on how our  existing machine may be
> retrofitted for this task.
>
> Thanks
>
> Peter Owens
>
>
> __________________________________
> Dr. Peter Owens
> Centre for Microscopy and Imaging and NBIPI
> National University of Ireland Galway
> Landline: 0035391494036
> Mobile:    [phone]
> ------------------------------------------------------
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Mark Cannell
> Sent: 02 May 2012 17:21
> To: [hidden email]
> Subject: Re: confocal PMT intermitently dies
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Check connectors...  Unplug and replug all connectors. If that doesn't
> work then its most likely electronic in the pmt3 power supply or pmt3
> preamp stages...
>
> Hope this helps
> Mark
>
> On 2/05/2012, at 4:22 PM, Arvydas Matiukas wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Dear list,
> >
> >
> > Ch3 PMT intermitently dies on our LSM510 system. After 2-4 hrs of
> > powering on, all image pixels turn to  zero intensity no matter what
> > are laser power, pinhole, filter, and gain settings are used.
> > Cold reboot does not fix the problem though no error messages are
> > reported. The single fix so far is to let the system cool down for
> > several hours then it is again working for few more hours.
> > Surprisingly all cooling fans are spinning normally as well as  room
> > conditioning system.
> >
> >
> >
> >
> > We are off service contract so any feedback/advice what could be wrong
>
> > and how to fix it is greatly appreciated.
> >
> >
> > Thank you,
> > Arvydas
> >
> >
> >
> >
> >
> > Arvydas Matiukas, Ph.D.
> > Director of Confocal&Two-Photon Imaging Core Facility Department of
> > Pharmacology SUNY Upstate Medical University
> > 766 Irving Ave., WH 3159
> > Syracuse, NY 13210
> > tel.: [phone]
> > fax: [phone]
> > email: [hidden email]
>

[Nuno Moreno]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Just one comment about NDDs. Please note that most SHG signals are forward...for a thin tissue. I also don't think it worth the investment.

Cheers,
Nuno Moreno

PS: don't forget http://uic.igc.gulbenkian.pt/coreworkshop.htm

On May 3, 2012, at 6:51 PM, Pascal Weber wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> What kinds of comments you want? how to align the IR laser!
> What will make the SHG? To align the IR laser desserts you a visible laser
> reflection in a mirror you will see out through the IR input beam visible. All you
> need to align two pinholes and convey your IR laser by two holes. For the SHG
> detectors you can use the simple but their performance will be low. The SHG is
> always half the wavelength of excitation. The best way is ti use  NDD

[Craig Brideau]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

For thick samples like tissue sections you will actually get significant
backscatter and it will work quite well.  On the other hand if you are
trying to do this on cell monolayers it will be very weak.

Craig


On Thu, May 3, 2012 at 12:26 PM, Nuno Moreno <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Just one comment about NDDs. Please note that most SHG signals are
> forward...for a thin tissue. I also don't think it worth the investment.
>
> Cheers,
> Nuno Moreno
>
> PS: don't forget http://uic.igc.gulbenkian.pt/coreworkshop.htm
>
> On May 3, 2012, at 6:51 PM, Pascal Weber wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > What kinds of comments you want? how to align the IR laser!
> > What will make the SHG? To align the IR laser desserts you a visible
> laser
> > reflection in a mirror you will see out through the IR input beam
> visible. All you
> > need to align two pinholes and convey your IR laser by two holes. For
> the SHG
> > detectors you can use the simple but their performance will be low. The
> SHG is
> > always half the wavelength of excitation. The best way is ti use  NDD
>

[Pascal Weber]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

The most important signal in SHG is always straight in the axis of the
excitation. But you can make the SHG as the classic flourescence, less signal
but it works.

[Steffen Dietzel]

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A bit late, but anyway a comment on the forward/backward issue:
We could significantly improve the intensity of the SHG (and THG) signal
recorded by the backward-detector by putting a mirror under a thin
sample (150 - 200 um thick muscle). A few cents worth of hardware
resulted in a 8-10x brighter signal.
See
http://spiedigitallibrary.org/jbo/resource/1/jbopfo/v15/i2/p026017_s1

cheers
Steffen

On 03.05.2012 20:26, Nuno Moreno wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Just one comment about NDDs. Please note that most SHG signals are forward...for a thin tissue. I also don't think it worth the investment.
>
> Cheers,
> Nuno Moreno
>
> PS: don't forget http://uic.igc.gulbenkian.pt/coreworkshop.htm
>
> On May 3, 2012, at 6:51 PM, Pascal Weber wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> What kinds of comments you want? how to align the IR laser!
>> What will make the SHG? To align the IR laser desserts you a visible laser
>> reflection in a mirror you will see out through the IR input beam visible. All you
>> need to align two pinholes and convey your IR laser by two holes. For the SHG
>> detectors you can use the simple but their performance will be low. The SHG is
>> always half the wavelength of excitation. The best way is ti use  NDD

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

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